Optoinjection methods

ABSTRACT

Optoinjection method for transiently permeabilizing a target cell by (a) illuminating a population of cells contained in a frame; (b) detecting at least one property of light directed from the frame; (c) locating a target cell by the property of light; and (d) irradiating the target cell with a pulse of radiation.

[0001] This application is a continuation in part of U.S. patentapplication Ser. No. 09/728,281, filed Nov. 30, 2000; which is acontinuation in part of application Ser. No. 09/451,659, filed Nov. 30,1999; which is a continuation in part of application Ser. No.09/049,677, filed Mar. 27, 1998, now U.S. Pat. No. 6,143,535; which is acontinuation in part of application Ser. No. 08/824,968, filed Mar. 27,1997, now U.S. Pat. No. 5,874,266, each of which is incorporated byreference herein.

BACKGROUND OF THE INVENTION

[0002] This invention relates to methods for cell manipulation and morespecifically to methods for transiently permeabilizing a cell so that avariety of exogenous materials, such as expressible foreign DNA, can beloaded into the cell.

[0003] Previous loading methods have included chemical treatments,microinjection, electroporation and particle bombardment. However, thesetechniques can be time-consuming and suffer from low yields or poor cellsurvival. Another technique termed “optoporation” has used lightdirected toward cells and the surrounding media to induce shock waves,thereby causing small holes to form temporarily in the surface of nearbycells, allowing materials to non-specifically enter cells in the area.Another technique termed “optoinjection” also uses light, but directsthe light to specific cells. Nevertheless, previous light-basedimplementations techniques have suffered from the same disadvantages asother loading techniques.

[0004] Thus, there is a need for a method for rapid and efficientloading of a variety of exogenous molecules into cells, with high cellsurvival rates. The present invention satisfies this need and providesrelated advantages as well.

SUMMARY OF THE INVENTION

[0005] The present invention provides optoinjection methods fortransiently permeabilizing a target cell. In the general method, thesteps are (a) illuminating a population of cells contained in a frame;(b) detecting at least one property of light directed from the frame;(c) locating a target cell by the property of light; and (d) irradiatingthe target cell with a pulse of radiation.

[0006] In particular embodiments, a static representation is obtainedwhen the population of cells is substantially stationary; the cells areilluminated through a lens having a numerical aperture of at most 0.5;the pulse of radiation has a diameter of at least 10 microns at thepoint of contact with the target cell; or the resulting pulse ofradiation delivers at most 1 μJ/μm². As a result, the method providesrapid and efficient loading of a variety of exogenous molecules intocells, with high cell survival rates.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007]FIG. 1 is a perspective view of one embodiment of a cell treatmentapparatus and illustrates the outer design of the housing and display.

[0008]FIG. 2 is a perspective view of one embodiment of a cell treatmentapparatus with the outer housing removed and the inner componentsillustrated.

[0009]FIG. 3 is a block diagram of the optical subassembly design withinone embodiment of a cell treatment apparatus.

[0010]FIG. 4 is a perspective view of one embodiment of an opticalsubassembly within one embodiment of a cell treatment apparatus.

[0011]FIG. 5 is a side view of one embodiment of an optical subassemblythat illustrates the arrangement of the scanning lens and the movablestage.

[0012]FIG. 6 is a bottom perspective view of one embodiment of anoptical subassembly.

[0013]FIG. 7 is a top perspective view of the movable stage of the celltreatment apparatus.

[0014]FIG. 8 shows cells under broad-spectrum light (8A), cells showingloading of Texas-Red-Dextran (8B) and nonviable cells (8C).

[0015]FIG. 9 illustrates that the efficiency of optoinjection is energydose-dependent

[0016]FIG. 10 compares expression of a plasmid in optoinjected cells(10B) compared to control cells without optoinjection (10A).

DETAILED DESCRIPTION

[0017] A method and apparatus is described for selectively identifying,and individually targeting with an energy beam, specific cells within acell population for the purpose of inducing a response in the targetedcells. The population of cells can be a mixed population or homogenousin origin. The responses of any of the embodiments of the methods andapparatuses of the invention can be lethal or non-lethal. Examples ofsuch responses are set forth above and throughout this disclosure. Thecells targeted can be labeled as is often the case when the specimen isa mixed population. On the other hand, when the specimen is homogenous,the targeted cells can be those individual cells that are beinginterrogated or intersected by the illumination source or the energybeam, in order to study the response of the cell. For instance, suchresponses include the morphological or physiological characteristics ofthe cell. Generally, the method first employs a label that acts as amarker to identify and locate individual cells of a first population ofcells within a cell mixture that is comprised of the first population ofcells and a second population of cells. The cells targeted by theapparatus and methods herein are those that are selectively labeled, inthe case of a mixed population of cells, or the ones undergoinginterrogation or intersection by the illumination source or energy beam.

[0018] The chosen label can be any that substantially identifies anddistinguishes the first population of cells from the second populationof cells. For example, monoclonal antibodies that are directly orindirectly tagged with a fluorochrome can be used as specific labels.Other examples of cell surface binding labels include non-antibodyproteins, lectins, carbohydrates, or short peptides with selective cellbinding capacity. Membrane intercalating dyes, such as PKH-2 and PKH-26,could also serve as a useful distinguishing label indicating mitotichistory of a cell. Many membrane-permeable reagents are also availableto distinguish living cells from one another based upon selectedcriteria. For example, phalloidin indicates membrane integrity,tetramethyl rhodamine methyl ester (TMRM) indicates mitochondrialtransmembrane potential, monochlorobimane indicates glutathionereductive stage, carboxymethyl fluorescein diacetate (CMFDA) indicatesthiol activity, carboxyfluorescein diacetate indicates intracellular pH,fura-2 indicates intracellular Ca²⁺ level, and5,5′,6,6′-tetrachloro-1,1′1,3,3′-tetraethylbenzimidazolo carbocyanineiodide (JC-1) indicates membrane potential. Cell viability can beassessed by the use of fluorescent SYTO 13 or YO PRO reagents.Similarly, a fluorescently-tagged genetic probe (DNA or RNA) could beused to label cells which carry a gene of interest, or express a gene ofinterest. Further, cell cycle status could be assessed through the useof Hoechst 33342 dye to label existing DNA combined withbromodeoxyuridine (BrdU) to label newly synthesized DNA.

[0019] It should be noted that if no specific label is available forcells of the first population, the method can be implemented in aninverse fashion by utilizing a specific label for cells of the secondpopulation. For example, in hematopoietic cell populations, the CD34 orACC-133 cell markers can be used to label only the primitivehematopoietic cells, but not the other cells within the mixture. In thisembodiment, cells of the first population are identified by the absenceof the label, and are thereby targeted by the energy beam.

[0020] After cells of the first population are identified, an energybeam, such as from a laser, collimated or focused non-laser light, RFenergy, accelerated particle, focused ultrasonic energy, electron beam,or other radiation beam, is used to deliver a targeted dose of energythat induces the pre-determined response in each of the cells of thefirst population, without substantially affecting cells of the secondpopulation.

[0021] One such pre-determined response is photobleaching. Inphotobleaching, a label in the form of a dye, such as rhodamine 123,GFP, fluorescein isothiocyanate (FITC), or phycoerythrin, is added tothe specimen before the instant methods are commenced. After thepopulation of cells has time to interact with the dye, the energy beamis used to bleach a region of individual cells in the population. Suchphotobleaching studies can be used to study the motility, replenishment,dynamics and the like of cellular components and processes.

[0022] Another response is internal molecular uncaging. In such aprocess, the specimen is combined with a caged molecule prior to thecommencement of the instant methods. Such caged molecules include theβ-2,6-dinitrobenzyl ester of L-aspartic acid or the1-(2-nitrophenyl)ethyl ether of 8-hydroxylpyrene-1,3,6-tris-sulfonicacid. Similarly, caging groups including alphacarboxyl-2-nitrobenzyl(CNB) and 5-carboxylmethoxy-2-nitrobenzyl (CMNB) can be linked tobiologically active molecules as ethers, thioethers, esters, amines, orsimilar functional groups. The term “internal molecular uncaging” refersto the fact that the molecular uncaging takes place on the surface orwithin the cell. Such uncaging experiments study rapid molecularprocesses sucn as cell membrane permeability and cellular signaling.

[0023] Yet another response is external molecular uncaging. This usesapproximately the same process as internal molecular caging. However, inexternal molecular uncaging, the uncaged molecule is not attached to orincorporated into the targeted cells. Instead, the responses of thesurrounding targeted cells to the caged and uncaged variants of themolecule are imaged by the instant apparatus and methods.

[0024]FIG. 1 is an illustration of one embodiment of a cell treatmentapparatus 10. The cell treatment apparatus 10 includes a housing 15 thatstores the inner components of the apparatus. The housing includes lasersafety interlocks to ensure safety of the user, and also limitsinterference by external influences (e.g., ambient light, dust, etc.).Located on the upper portion of the housing 15 is a display unit 20 fordisplaying captured images of cell populations during treatment. Theseimages are captured by a camera, as will be discussed more specificallybelow. A keyboard 25 and mouse 30 are used to input data and control theapparatus 10. An access door 35 provides access to a movable stage thatholds a specimen container of cells undergoing treatment.

[0025] An interior view of the apparatus 10 is provided in FIG. 2. Asillustrated, the apparatus 10 provides an upper tray 200 and lower tray210 that hold the interior components of the apparatus. The upper tray200 includes a pair of intake filters 215A,B that filter ambient airbeing drawn into the interior of the apparatus 10. Below the access door35 is the optical subassembly (not shown). The optical subassembly ismounted to the upper tray 200 and is discussed in detail with regard toFIGS. 3 to 6.

[0026] On the lower tray 210 is a computer 225 which stores the softwareprograms, commands and instructions that run the apparatus 10. Inaddition, the computer 225 provides control signals to the treatmentapparatus through electrical signal connections for steering the laserto the appropriate spot on the specimen in order to treat the cells.

[0027] As illustrated, a series of power supplies 230A,B,C provide powerto the various electrical components within the apparatus 10. Inaddition, an uninterruptable power supply 235 is incorporated to allowthe apparatus to continue functioning through short external powerinterruptions.

[0028]FIG. 3 provides a layout of one embodiment of an opticalsubassembly design 300 within an embodiment of a cell treatmentapparatus 10. As illustrated, an illumination laser 305 provides adirected laser output that is used to excite a particular label that isattached to targeted cells within the specimen. In this embodiment, theillumination laser emits light at a wavelength of 532 nm. Once theillumination laser has generated a light beam, the light passes into ashutter 310 which controls the pulse length of the laser light.

[0029] After the illumination laser light passes through the shutter310, it enters a ball lens 315 where it is focused into a SMA fiberoptic connector 320. After the illumination laser beam has entered thefiber optic connector 320, it is transmitted through a fiber optic cable325 to an outlet 330. By passing the illumination beam through the fiberoptic cable 325, the illumination laser 305 can be positioned anywherewithin the treatment apparatus and thus is not limited to only beingpositioned within a direct light pathway to the optical components. Inone embodiment, the fiber optic cable 325 is connected to a vibratingmotor 327 for the purpose of mode scrambling and generating a moreuniform illumination spot.

[0030] After the light passes through the outlet 330, it is directedinto a series of condensing lenses in order to focus the beam to theproper diameter for illuminating one frame of cells. As used herein, oneframe of cells is defined as the portion of the biological specimen thatis captured within one frame image captured by the camera. This isdescribed more specifically below.

[0031] Accordingly, the illumination laser beam passes through a firstcondenser lens 335. In one embodiment, this first lens has a focallength of 4.6 mm. The light beam then passes through a second condenserlens 340 which, in one embodiment, provides a 100 mm focal length.Finally, the light beam passes into a third condenser lens 345, whichpreferably provides a 200 mm focal length. While the present inventionhas been described using specific condenser lenses, it should beapparent that other similar lens configurations that focus theillumination laser beam to an advantageous diameter would functionsimilarly. Thus, this invention is not limited to the specificimplementation of any particular condenser lens system.

[0032] Once the illumination laser beam passes through the thirdcondenser lens 345, it enters a cube beam splitter 350 that is designedto transmit the 532 nm wavelength of light emanating from theillumination laser. Preferably, the cube beam splitter 350 is a 25.4 mmsquare cube (Melles-Griot, Irvine, Calif.). However, other sizes areanticipated to function similarly. In addition, a number of plate beamsplitters or pellicle beam splitters could be used in place of the cubebeam splitter 350 with no appreciable change in function.

[0033] Once the illumination laser light has been transmitted throughthe cube beam splitter 350, it reaches a long wave pass mirror 355 thatreflects the 532 nm illumination laser light to a set of galvanometermirrors 360 that steer the illumination laser light under computercontrol to a scanning lens (Special Optics, Wharton, N.J.) 365, whichdirects the illumination laser light to the specimen (not shown). Thegalvanometer mirrors are controlled so that the illumination laser lightis directed at the proper cell population (i.e. frame of cells) forimaging. The “scanning lens” described in this embodiment of theinvention includes a refractive lens. It should be noted that the term“scanning lens” as used in the present invention includes, but is notlimited to, a system of one or more refractive or reflective opticalelements used alone or in combination. Further, the “scanning lens” mayinclude a system of one or more diffractive elements used in combinationwith one or more refractive and/or reflective optical elements. Oneskilled in the art will know how to design a “scanning lens” system inorder to illuminate the proper cell population.

[0034] The light from the illumination laser is of a wavelength that isuseful for illuminating the specimen. In this embodiment, energy from acontinuous wave 532 nm Nd:YAG frequency-doubled laser (B&W Tek, Newark,Del.) reflects off the long wave pass mirror (Custom Scientific,Phoenix, Ariz.) and excites fluorescent tags in the specimen. In oneembodiment, the fluorescent tag is phycoerythrin. Alternatively, Alexa532 (Molecular Probes, Eugene, Oreg.) can be used. Phycoerythrin andAlexa 532 have emission spectra with peaks near 580 nm, so that theemitted fluorescent light from the specimen is transmitted via the longwave pass mirror to be directed into the camera. The use of the filterin front of the camera blocks light that is not within the wavelengthrange of interest, thereby reducing the amount of background lightentering the camera.

[0035] It is generally known that many other devices could be used inthis manner to illuminate the specimen, including, but not limited to,an arc lamp (e.g., mercury, xenon, etc.) with or without filters, alight-emitting diode (LED), other types of lasers, etc. Advantages ofthis particular laser include high intensity, relatively efficient useof energy, compact size, and minimal heat generation. It is alsogenerally known that other fluorochromes with different excitation andemission spectra could be used in such an apparatus with the appropriateselection of illumination source, filters, and long and/or short wavepass mirrors. For example, allophycocyanin (APC) could be excited with a633 nm HeNe illumination laser, and fluoroisothiocyanate (FITC) could beexcited with a 488 nm Argon illumination laser. One skilled in the artcould propose many other optical layouts with various components inorder to achieve the objective of this invention.

[0036] In addition to the illumination laser 305, an optional treatmentlaser 400 is present to irradiate the targeted cells once they have beenidentified by image analysis. Of course, in one embodiment, thetreatment induces necrosis of targeted cells within the cell population.As shown, the treatment laser 400 outputs an energy beam of 523 nm thatpasses through a shutter 410. Although the exemplary laser outputs anenergy beam having a 523 nm wavelength, other sources that generateenergy at other wavelengths are also within the scope or the presentinvention.

[0037] Once the treatment laser energy beam passes through the shutter410, it enters a beam expander (Special Optics, Wharton, N.J.) 415 whichadjusts the diameter of the energy beam to an appropriate size at theplane of the specimen. Following the beam expander 415 is a half-waveplate 420 which controls the polarization of the beam. The treatmentlaser energy beam is then reflected off a mirror 425 and enters the cubebeam splitter 350. The treatment laser energy beam is reflected by 90degrees in the cube beam splitter 350, such that it is aligned with theexit pathway of the illumination laser light beam. Thus, the treatmentlaser energy beam and the illumination laser light beam both exit thecube beam splitter 350 along the same light path. From the cube beamsplitter 350, the treatment laser beam reflects off the long wave passmirror 355, is steered by the galvanometers 360, thereafter contacts thescanning lens 365, and finally is focused upon a targeted cell withinthe specimen. Again, the “scanning lens” described in this embodimentincludes a refractive lens. As previously mentioned, the term “scanninglens” includes, but is not limited to, a system of one or morerefractive or reflective optical elements used alone or in combination.Further, the “scanning lens” may include one or more diffractiveelements used in combination with one or more refractive and/orreflective elements. One skilled in the art will know how to design a“scanning lens” system in order to focus upon the targeted cell withinthe specimen.

[0038] It should be noted that a small fraction of the illuminationlaser light beam passes through the long wave pass mirror 355 and entersa power meter sensor (Gentec, Palo Alto, Calif.) 445. The fraction ofthe beam entering the power sensor 445 is used to calculate the level ofpower emanating from the illumination laser 305. In an analogousfashion, a small fraction of the treatment laser energy beam passesthrough the cube beam splitter 350 and enters a second power metersensor (Gentec, Palo Alto, Calif.) 446. The fraction of the beamentering the power sensor 446 is used to calculate the level of poweremanating from the treatment laser 400. The power meter sensors areelectrically linked to the computer system so that instructions/commandswithin the computer system capture the power measurement and determinethe amount of energy that was emitted.

[0039] The energy beam from the treatment laser is of a wavelength thatis useful for achieving a response in the cells. In the example shown, apulsed 523 nm Nd:YLF frequency-doubled laser is used to heat a localizedvolume containing the targeted cell, such that it is induced to diewithin a pre-determined period of time. The mechanism of death isdependent upon the actual temperature achieved in the cell, as reviewedby Niemz, M. H., Laser-tissue interactions: Fundamentals andApplications (Springer-Verlag, Berlin 1996).

[0040] A Nd:YLF frequency-doubled, solid-state laser (Spectra-Physics,Mountain View, Calif.) is used because of its stability, high repetitionrate of firing, and long time of maintenance-free service. However, mostcell culture fluids and cells are relatively transparent to light inthis green wavelength, and therefore a very high fluence of energy wouldbe required to achieve cell death. To significantly reduce the amount ofenergy required, and therefore the cost and size of the treatment laser,a dye is purposefully added to the specimen to efficiently absorb theenergy of the treatment laser in the specimen. In the example shown, thenon-toxic dye FD&C red #40 (allura red) is used to absorb the 523 nmenergy from the treatment laser, but one skilled in the art couldidentify other laser/dye combinations that would result in efficientabsorption of energy by the specimen. For example, a 633 nm HeNe laser'senergy would be efficiently absorbed by FD&C green #3 (fast green FCF),a 488 nm Argon laser's energy would be efficiently absorbed by FD&Cyellow #5 (sunset yellow FCF), and a 1064 nm Nd:YAG laser's energy wouldbe efficiently absorbed by Filtron (Gentex, Zeeland, Mich.) infraredabsorbing dye. Through the use of an energy absorbing dye, the amount ofenergy required to kill a targeted cell can be reduced since more of thetreatment laser energy is absorbed in the presence of such a dye.

[0041] Another method of achieving thermal killing of cells without theaddition of a dye involves the use of an ultraviolet laser. Energy froma 355 nm Nd:YAG frequency-tripled laser will be absorbed by nucleicacids and proteins within the cell, resulting in thermal heating anddeath. Yet another method of achieving thermal killing of cells withoutthe addition of a dye involves the use of a near-infrared laser. Energyfrom a 2100 nm Ho:YAG laser or a 2940 nm Er:YAG laser will be absorbedby water within the cell, resulting in thermal heating and death.

[0042] Although this embodiment describes the killing of cells viathermal heating by the energy beam, one skilled in the art wouldrecognize that other responses can also be induced in the cells by anenergy beam, including photomechanical disruption, photodissociation,photoablation, and photochemical reactions, as reviewed by Niemz (Niemz,supra). For example, a photosensitive substance (e.g., hematoporphyrinderivative, tinetiopurpurin, lutetium texaphyrin) (Oleinick and Evans,The photobiology of photodynamic therapy: Cellular targets andmechanisms, Rad. Res. 150: S146-S156 (1998)) within the cell mixturecould be specifically activated in targeted cells by irradiation.Additionally, a small, transient pore could be made in the cell membrane(Palumbo et al., Targeted gene transfer in eukaryotic cells bydye-assisted laser optoporation, J. Photochem. Photobiol. 36:41-46(1996)) to allow the entry of genetic or other material. Further,specific molecules in or on the cell, such as proteins or geneticmaterial, could be inactivated bv the directed energy beam (Grate andWilson, Laser-mediated, site-specific inactivation of RNA transcripts,PNAS 96:6131-6136 (1999); Jay, D. G., Selective destruction of proteinfunction by chromophore-assisted laser inactivation, PNAS 85:5454-5458(1988)). Also, photobleaching can be utilized to measure intracellularmovements such as the diffusion of proteins in membranes and themovements of microtubules during mitosis (Ladha et al., J. Cell Sci.,110(9):1041 (1997); Centonze and Borisy, J. Cell Sci. 100 (part 1):205(1991); White and Stelzer, Trends Cell Biol. 9(2):61-5 (1999); Meyvis,et al., Pharm. Res. 16(8):1153-62 (1999). Further, photolysis oruncaging, including multiphoton uncaging, of caged compounds can beutilized to control the release, with temporal and spacial resolution,of biologically active products or other products of interest (Theriotand Mitchison, J. Cell Biol. 119:367 (1992); Denk, PNAS 91(14):6629(1994)). These mechanisms of inducing a response in a targeted cell viathe use of electromagnetic radiation directed at specific targeted cellsare also intended to be incorporated into the present invention.

[0043] In addition to the illumination laser 305 and treatment laser400, the apparatus includes a camera 450 that captures images (i.e.frames) of the cell populations. As illustrated in FIG. 3, the camera450 is focused through a lens 455 and filter 460 in order to accuratelyrecord an image of the cells without capturing stray background images.A stop 462 is positioned between the filter 460 and mirror 355 in orderto eliminate light that may enter the camera from angles not associatedwith the image from the specimen. The filter 460 is chosen to only allowpassage of light within a certain wavelength range. This wavelengthrange includes light that is emitted from the targeted cells uponexcitation by the illumination laser 305, as well as light from aback-light source 475.

[0044] The back-light source 475 is located above the specimen toprovide back-illumination of the specimen at a wavelength different fromthat provided by the illumination laser 303. This LED generates light at590 nm, such that it can be transmitted through the long wave passmirror to be directed into the camera. This back-illumination is usefulfor imaging cells when there are no fluorescent targets within the framebeing imaged. An example of the utility of this back-light is its use inattaining proper focus of the system, even when there are onlyunstained, non-fluorescent cells in the frame. In one embodiment, theback-light is mounted on the underside of the access door 35 (FIG. 2).

[0045] Thus, as discussed above, the only light returned to the camerais from wavelengths that are of interest in the specimen. Otherwavelengths of light do not pass through the filter 460, and thus do notbecome recorded by the camera 450. This provides a more reliablemechanism for capturing images of only those cells of interest. It isreadily apparent to one skilled in the art that the single filter 460could be replaced by a movable filter wheel that would allow differentfilters to be moved in and out of the optical pathway. In such anembodiment, images of different wavelengths of light could be capturedat different times during cell processing, allowing the use of multiplecell labels.

[0046] It should be noted that in this embodiment, the camera is acharge-coupled device (CCD) and transmits images back to the computersystem for processing. As will be described below, the computer systemdetermines the coordinates of the targeted cells in the specimen byreference to the image captured by the CCD camera.

[0047] Referring now to FIG. 4, a perspective view of an embodiment ofan ootical subassembly is illustrated. As illustrated, the illuminationlaser 305 sends a light beam through the shutter 310 and ball lens 315to the SMA fiber optic connector 320. The light passes through the fiberoptic cable 325 and through the output 330 into the condenser lenses335, 340 and 345. The light then enters the cube beam splitter 350 andis transmitted to the long wave pass mirror 355. From the long wave passmirror 355, the light beam enters the computer-controlled galvanometers360 and is then steered to the proper frame of cells in the specimenfrom the scanning lens 365.

[0048] As also illustrated in the perspective drawing of FIG. 4, thetreatment laser 400 transmits energy through the shutter 410 and intothe beam expander 415. Energy from the treatment laser 400 passesthrough the beam expander 415 and passes thrbugh the half-wave plate 420before hitting the fold mirror 425, entering the cube beam splitter 350where it is reflected 90 degrees to the long wave pass mirror 355, fromwhich it is reflected into the computer controlled galvanometer mirrors360. After being steered by the galvanometer mirrors 360 to the scanninglens 365, the laser energy beam strikes the proper location within thecell population in order to induce a response in a particular targetedcell.

[0049] In order to accommodate a very large surface area of specimen totreat, the apparatus includes a movable stage that mechanically movesthe specimen container with respect to the scanning lens. Thus, once aspecific sub-population (i.e. field) of cells within the scanning lensfield-of-view has been treated, the movable stage brings anothersub-population of cells within the scanning lens field-of-view. Asillustrated in FIG. 5, a computer-controlled movable stage 500 holds aspecimen container (not shown) to be processed. The movable stage 500 ismoved by computer-controlled servo motors along two axes so that thespecimen container can be moved relative to the optical components ofthe instrument. The stage movement along a defined path is coordinatedwith other operations of the apparatus. In addition, specificcoordinates can be saved and recalled to allow return of the movablestage to positions of interest. Encoders on the x and y movement provideclosed-loop feedback control on stage position.

[0050] The flat-field (F-theta) scanning lens 365 is mounted below themovable stage. The scanning lens. field-of-view comprises the portion ofthe specimen that is presently positioned above the scanning lens by themovable stage 500. The lens 365 is mounted to a stepper motor thatallows the lens 365 to be automatically raised and lowered (along thez-axis) for the purpose of focusing the system.

[0051] As illustrated in FIGS. 4 to 6, below the scanning lens 365 arethe galvanometer-controlled steering mirrors 360 that deflectelectromagnetic energy along two perpendicular axes. Behind the steeringmirrors is the long wave pass mirror 355 that reflects electromagneticenergy of a wavelength shorter than 545 nm. Wavelengths longer than 545nm are passed through the long wave pass mirror, directed through thefilter 460, coupling lens 455, and into the CCD camera, therebyproducing an image of the appropriate size on the CCD sensor of thecamera 450 (see FIGS. 3 and 4). The magnification defined by thecombination of the scanning lens 365 and coupling lens 455 is chosen toreliably detect single cells while maximizing the area viewed in oneframe by the camera. Although a CCD camera (DVC, Austin, Tex.) isillustrated in this embodiment, the camera can be any type of detectoror image gathering equipment known to those skilled in the art. Theoptical subassembly of the apparatus is preferably mounted on avibration-isolated platform to provide stability during operation asillustrated in FIGS. 2 and 5.

[0052] Referring now to FIG. 7, a top view of the movable stage 500 isillustrated. As shown, a specimen container is mounted in the movablestage 500. The specimen container 505 rests on an upper axis nest plate510 that is designed to move in the forward/backward direction withrespect to the movable stage 500. A stepper motor (not shown) isconnected to the upper axis nest plate 510 and computer system so thatcommands from the computer cause forward/backward movement of thespecimen container 505.

[0053] The movable stage 500 is also connected to a timing belt 515 thatprovides side-to-side movement of the movable stage 500 along a pair ofbearing tracks 525A,B. The timing belt 515 attaches to a pulley (notshown) housed under a pulley cover 530. The pulley is connected to astepper motor 535 that drives the timing belt 515 to result inside-to-side movement of the movable stage 500. The stepper motor 535 iselectrically connected to the computer system so that commands withinthe computer system result in side-to-side movement of the movable stage500. A travel limit sensor 540 connects to the computer system andcauses an alert if the movable stage travels beyond a predeterminedlateral distance.

[0054] A pair of accelerometers 545A,B is preferably incorporated onthis platform to register any excessive bumps or vibrations that mayinterfere with the apparatus operation. In addition, a two-axisinclinometer 550 is preferably incorporated on the movable stage toensure that the specimen container is level, thereby reducing thepossibility of gravity-induced motion in the specimen container.

[0055] The specimen chamber has a fan with ductwork to eliminatecondensation on the specimen container, and a thermocouple to determinewhether the specimen chamber is within an acceptable temperature range.Additional fans are provided to expel the heat generated by theelectronic components, and appropriate filters are used on the airintakes 215A,B.

[0056] The computer system 225 controls the operation andsynchronization of the various pieces of electronic hardware describedabove. The computer system can be any commercially available computerthat can interface with the hardware. One example of such a computersystem is an Intel Pentium II, III or IV-based computer running theMicrosoft WINDOWS NT operating system. Software is used to communicatewith the various devices, and control the operation in the manner thatis described below.

[0057] When the apparatus is first initialized, the computer loads filesfrom the hard drive into RAM for proper initialization of the apparatus.A number of built-in tests are automatically performed to ensure theapparatus is operating properly, and calibration routines are executedto calibrate the apparatus. Upon successful completion of theseroutines, the user is prompted to enter information via the keyboard andmouse regarding the procedure that is to be performed. Once the requiredinformation is entered, the user is prompted to open the access door 35and load a specimen onto the movable stage.

[0058] Once a specimen is in place on the movable stage and the door isclosed, the computer passes a signal to the stage to move into a homeposition. The fan is initialized to begin warming and defogging of thespecimen. During this time, cells within the specimen are allowed tosettle to the bottom surface. In addition, during this time, theapparatus may run commands that ensure that the specimen is properlyloaded, and is within the focal range of the system optics. For example,specific markings on the specimen container can be located and focusedon by the system to ensure that the scanning lens has been properlyfocused on the bottom of the specimen container. Such markings couldalso be used by the instrument to identify the container, its contents,and even the procedure to be performed. After a suitable time, thecomputer turns off the fan to prevent excess vibrations duringtreatment, and cell processing begins.

[0059] First, the computer instructs the movable stage to be positionedover the scanning lens so that the first area (i.e. field) of thespecimen to be treated is directly in the scanning lens field-of-view.The galvanometer mirrors are instructed to move such that the centerframe within the field-of-view is imaged in the camera. As discussedbelow, the field imaged by the scanning lens is separated into aplurality of frames. Each frame is the proper size so that the cellswithin the frame are effectively imaged by the camera.

[0060] The back-light 475 is then activated in order to illuminate thefield-of-view so that it can be brought into focus by the scanning lens.Once the scanning lens has been properly focused upon the specimen, thecomputer system divides the field-of-view into a plurality of frames sothat each frame is analyzed separately by the camera. This methodologyallows the apparatus to process a plurality of frames within a largefield-of-view without moving the mechanical stage. Because thegalvanometers can move from one frame to the next very rapidly comparedto the mechanical steps involved in moving the stage, this methodresults is an extremely fast and efficient apparatus.

[0061] Other means of ensuring that the specimen is in focus are alsoavailable. For example, a laser proximeter (Cooke Corp., Auburn, Mich.)could rapidly determine the distance between the scanning lens and thesample, and adjust the scanning lens position accordingly. Ultrasonicproximeters are also available, and would achieve the same objective.One skilled in the art could propose other means of ensuring that thespecimen is in focus above the scanning lens. in one preferredembodiment, the apparatus described herein orocesses at least 1, 2, 3,4, 5, 6, 7, or 14 square centimeters of a biological specimen perminute. In another embodiment, the apparatus described herein processesat least 0.25, 0.5, 1, 2, 3, 4 or 8 million cells of a biologicalspecimen per minute. In one other embodiment, the apparatus canpreferably induce a response in targeted cells at a rate of 50, 100,150, 200, 250, 300, 350, 400 or 800 cells per second.

[0062] Initially, an image of the frame at the center of thefield-of-view is captured by the camera and stored to a memory in thecomputer. Instructions in the computer analyze the focus of the specimenby looking at the size of, number of, and other object features in theimage. If necessary, the computer instructs the z-axis motor attached tothe scanning lens to raise or lower in order to achieve the best focus.The apparatus may iteratively analyze the image at several z-positionsuntil the best focus is achieved. The galvanometer-controlled mirrorsare then instructed to image a first frame, within the field-of-view, inthe camera. For example, the entire field-of-view might be divided into4, 9, 12, 18, 24 or more separate frames that will be individuallycaptured by the camera. Once the galvanometer mirrors are pointed to thefirst frame in the field-of-view, the shutter in front of theillumination laser is opened to illuminate the first frame through thegalvanometer mirrors and scanning lens. The camera captures an image ofany fluorescent emission from the specimen in the first frame of cells.Once the image has been acquired, the shutter in front of theillumination laser is closed and a software program (Epic, BuffaloGrove, Ill.) within the computer processes the image.

[0063] The power sensor 445 discussed above detects the level of lightthat was emitted by the illumination laser, thereby allowing thecomputer to calculate if it was adequate to illuminate the frame ofcells. If not, another illumination and image capture sequence isperformed. Repeated failure to sufficiently illuminate the specimen willresult in an error condition that is communicated to the operator.

[0064] Shuttering of illumination light reduces undesirable heating andphotobleaching of the specimen and provides a more repeatablefluorescent signal. An image analysis algorithm is run to locate the x-ycentroid coordinates of all targeted cells in the frame by reference tofeatures in the captured image. If there are targets in the image, thecomputer calculates the two-dimensional coordinates of all targetlocations in relation to the movable stage position and field-of-view,and then positions the galvanometer-controlled mirrors to point to thelocation of the first target in the first frame of cells. It should benoted that only a single frame of cells within the field-of-view hasbeen captured and analyzed at this point. Thus, there should be arelatively small number of identified targets within this sub-populationof the specimen. Moreover, because the camera is pointed to a smallerpopulation of cells, a higher magnification is used so that each targetis imaged by many pixels within the CCD camera.

[0065] Once the computer system has positioned the galvanometercontrolled mirrors to point to the location of the first targeted cellwithin the first frame of cells, the treatment laser is fired for abrief interval so that the first targeted cell is given an appropriatedose of energy. The power sensor 446 discussed above detects the levelof energy that was emitted by the treatment laser, thereby allowing thecomputer to calculate if it was adequate to induce a response in thetargeted cell. If not sufficient, the treatment laser is fired at thesame target again. If repeated shots do not deliver the required energydose, an error condition is communicated to the operator. Thesetargeting, firing, and sensing steps are repeated by the computer forall targets identified in the captured frame.

[0066] Once all of the targets have been irradiated with the treatmentlaser in the first frame of cells, the mirrors are then positioned tothe second frame of cells in the field-of-view, and the processingrepeats at the point of frame illumination and camera imaging. Thisprocessing continues for all frames within the field-of-view above thescanning lens. When all of these frames have been processed, thecomputer instructs the movable stage to move to the next field-of-viewin the specimen, and the process repeats at the back-light illuminationand auto-focus step. Frames and fields-of-view are appropriatelyoverlapped to reduce the possibility of inadvertently missing areas ofthe specimen. Once the specimen has been fully processed, the operatoris signaled to remove the specimen, and the apparatus is immediatelyready for the next specimen.

[0067] Although the text above describes the analysis of fluorescentimages for locating targets, one can easily imagine that thenon-fluorescent back-light LED illumination imaces will be useful forlocating other types of targets as well, even if they are unlabeled.

[0068] The advantage of using the galvanometer mirrors to control theimaging of successive frames and the irradiation of successive targetsis significant. One brand of galvanometer is the Cambridge Technology,Inc. model number 6860 (Cambridge, Mass.). This galvanometer canreposition very accurately within a few milliseconds, making theprocessing of large areas and many targets possible within a reasonableamount of time. In contrast, the movable stage is relatively slow, andis therefore used only to move specified areas of the specimen into thescanning lens field-of-view. Error signals continuously generated by thegalvanometer control boards are monitored by the computer to ensure thatthe mirrors are in position and stable before an image is captured, orbefore a target is fired upon, in a closed-loop fashion.

[0069] In the context of the present invention, the term “specimen” hasa broad meaning. It is intended to encompass any type of biologicalsample placed within the apparatus. The specimen may be enclosed by, orassociated with, a container to maintain the sterility and viability ofthe cells. Further, the specimen may incorporate, or be associated with,a cooling apparatus to keep it above or below ambient temperature duringoperation of the methods described herein. The specimen container, ifone is used, must be compatible with the use of the illumination laser,back-light illuminator, and treatment laser, such that it transmitsadequate energy without being substantially damaged itself.

[0070] Of course, many variations of the above-described embodiment arepossible, including alternative methods for illuminating, imaging, andtargeting the cells. For example, movement of the specimen relative tothe scanning lens could be achieved by keeping the specimensubstantially stationary while the scanning lens is moved. Steering ofthe illumination beam, images, and energy beam could be achieved throughany controllable reflective or diffractive device, including prisms,piezo-electric tilt platforms, or acousto-optic deflectors.Additionally, the apparatus can image/process from either below or abovethe specimen. Because the apparatus is focused through a movablescanning lens, the illumination and energy beams can be directed todifferent focal planes along the z-axis. Thus, portions of the specimenthat are located at different vertical heights can be specificallyimaged and processed by the apparatus in a three-dimensional manner. Thesequence of the steps could also be altered without changing theprocess. For example, one might locate and store the coordinates of alltargets in the specimen, and then return to the targets to irradiatethem with energy one or more times over a period of time.

[0071] To optimally process the specimen, it should be placed on asubstantially flat surface so that a large portion of the specimenappears within a narrow range of focus, thereby reducing the need forrepeated auto-focus steps. The density of cells on this surface can, inprinciple, be at any value. However, the cell density should be as highas possible to minimize the total surface area required for theprocedure.

[0072] A further embodiment of the invention provides optoinjectionmethods for transiently permeabilizing a target cell. In the generalmethod, the steps are (a) illuminating a population of cells containedin a frame; (b) detecting at least one property of light directed fromthe frame; (c) locating a target cell by the property of light; and (d)irradiating the target cell with a pulse of radiation.

[0073] The “cells” used in the method can be any, biological cells,including procaryotic and eucaryotic cells, such as animal cells, plantcells, yeast cells, human cells and non-human primate cells. The cellscan be taken from organisms or harvested from cell cultures. The methodcan also be applied to permeabilize subcellular organelles.

[0074] It follows that the term “population” of cells means a group ofmore than one of such cells. While performing the method, the populationof cells can be presented in a specimen container such as 505.

[0075] The cells can also be associated with an exogenous label such asa fluorophore. Other labels useful in the invention have been describedin detail above.

[0076] The population of cells can be “illuminated” by any source thatcan provide light energy, including a laser and an arc lamp. The lightenergy can be of any wavelength, such as visible, ultraviolet andinfrared light. When the light is from a laser, such as 400, usefulwavelengths can range from 100 nm to 1000 nm, 200 nm to 800 nm, 320 nmto 695 nm, and 330 nm to 605 nm. Particular wavelengths include 349 nm,355 nm, 488 nm, 523 nm, 532 nm, 580 nm, 590 nm, 633 nm, 1064 nm, 2100 nmand 2940 nm. Other illumination sources include any source for an energybeam, as described in detail above. The light can then be directed byany conventional means, such as mirrors, lenses and beam-splitters, tothe population of cells.

[0077] Once the cells are illuminated, they can be observed in a“frame.” As previously defined, one “frame” of cells is the portion ofthe biological specimen that is captured within one frame image capturedby the camera. A particularly useful frame can have an area of at least50, 70, 85, 95 or 115 mm². A useful magnification range for the camerais between 2× and 40× and more particularly between 2.5× and 25× andstill more particularly between 5× and 10×.

[0078] When the frame is illuminated, one or more properties of lightcan then be detected from the frame. The detectable properties includelight having visible, ultraviolet and infrared wavelengths, theintensity of transmittance and reflectance, fluorescence, linear andcircular polarization, and phase-contrast illumination. These propertiescan be detected by conventional optical devices such as the devicesalready described in detail above.

[0079] The target cell can then be located based on its size, shape andother preselected visual properties, and then irradiated with a pulse ofradiation. The radiation then causes a temporary permeabilization of thesurface of the target cell. While not limiting the method to aparticular mechanism, it is believed that the light causes localizedmelting or other disruption of the cell membrane's continuity, allowingsmall pores to form without killing the cell.

[0080] As a result of transiently permeabilizing the cells, exogenousmolecules in the presence of the cell can then enter the cell, whetherby diffusion or other mechanism. The term “presence of the cell” hereinas applied to an exogenous molecule means in the area near the cell,such as the surrounding medium, so that if the cell were permeabilized,the exogenous molecule could then enter the cell.

[0081] The term “exogenous molecule” herein means any molecule ormaterial that does not naturally occur in the cells of the population.It also includes molecules or materials that may occur naturally in thecell, but in significantly higher concentrations than occur naturally inthe cell. Exogenous molecules include nucleic acids, polypeotides,carbohydrates, lipids and small molecules. Particular nucleic acidsinclude RNAs, expression plasmids, expression cassettes and otherexpressible DNA. Particular polypeptides include antibodies and otherproteins, which can be introduced into cells to explore interactionsbetween exogenous and endogenous proteins for applications inproteomics. Other polypeptides include peptides for introduction intoantigen-displaying dendritic cells. Particular carbohydrates includenon-naturally occurring metabolites, such as isotopically labeledsugars, and polysaccharides, such as labeled dextrans. Particular lipidsinclude preselected lipids for incorporation into the cell membrane orother organelles, as well as liposomes and liposomes containing otherexogenous molecules of interest. Particular small molecules includeligands for endogenous receptors to study ligand-receotor binding.Similarly, drugs can be introduced into cells, which, in turn, can beintroduced as a delivery device into a patient for therapeutic purposes.The term also encompasses dyes capable of absorbing visible, ultravioletor infrared light.

[0082] Exogenous molecules can have a size of greater than 0.1, 0.2,0.3, 0.5, 1, 2, 3, 5, 10, 20, 30, 50, 70, 100 or even 200 kiloDaltons.Although the efficiencv rate of cells that are loaded with at least oneexogenous molecule will vary depending on the size and nature of theexogenous molecule, loading efficiencies can be as high as 5%, 10%, 20%,50%, 75% or even 90% of the population of cells. It should also beemphasized that the method encompasses techniques where two or moreexogenous molecules are loaded into cells simultaneously orsequentially.

[0083] Significantly, as result of using the method, greater than 50%,60%, 70%, 80%, 90%, 95% or even 98% of the irradiated target cells canbe viable after completion of the method. Methods for measuring cellsurvival rates are well known in the art and membrane-permeable reagentsfor distinguishing living cells have been described above. For example,preselected reagents can be added to the media before, during or afterperforming the method. Specific examples of useful reagents includeCalcein AM as an indicator of viability and Sytox Blue as an indicatorfor dead cells. Other well-known methods include trypan blue exclusion,propidium iodide and ⁵¹Cr-release assay.

[0084] It should be noted that the general method presented above hasseveral alternate embodiments that are particularly useful.

[0085] First, the general method can be used when the population ofcells is substantially stationary. The term “substantially stationary”herein means that the cells are relatively immobile with respect to themedium and are not in flowing medium, and the cells are not subjected togross movement of a container; but, they can be subject to vibrationsand slight movements that normally occur in a typical laboratory. Whilethe term encompasses cells that are immobilized to a surface or withinthe medium, substantially stationary cells need not be immobilized orotherwise bound to a surface to be considered substantially stationary.Thus; the term includes cells that have settled to the bottom of aspecimen container.

[0086] When a population of cells is substantially stationary, itbecomes useful to obtain a static representation of the cells in theframe. The term “static representation” herein means a substantiallycomplete image of the cells taken during a fixed and discrete timeperiod, rather than as a continuous image, as in a “live” monitor.

[0087] Because the cells are substantially stationary, the staticrepresentation can then be used as a reliable indicator of the locationof one or more cells at subsequent points in time. Moreover, a staticrepresentation can be obtained under one set of conditions and anotherstatic representation obtained under a different set of conditions sothat the two representations can be compared usefully without undueconcern for movement of the cells. For example, an image of the cellsunder visible light can be compared with a corresponding fluorescenceimage to identify fllorescently tagged cells of interest among a generalpopulation of cells. The static representation can also be used as thebasis for computer-aided identification and determination of thelocation of a target cell of interest, based on any of the lightproperties discussed above.

[0088] Second, the general method can be performed where the populationof cells is illuminated through a lens having numerical aperture of atmost 0.5, 0.4 or 0.3. The term “numerical aperture” or “N.A.” usedherein is defined N.A.=n(sin μ), where n is the refractive index of theimaging medium between the lens and the cells, and μ is one-half of theangular aperture.

[0089] As a consequence of using a lens having such a low numericalaperture, the lens can have a greater working distance, such as at least5, 7 or 10 mm. The term “working distance” herein means the distancebetween the front of the lens to the object, meaning the nearest surfaceof the population of cells. A particularly useful lens is a flat-field(F-theta) lens, as exemplified by lens 365, described above. It shouldbe noted that confocal microscopy is not possible under such lensparameters.

[0090] Third, the pulse of radiation can have a diameter of at least 2,5, 7, 10, 15, 20, 25 or 30 microns at the point of contact with thetarget cell. In most cases, the breadth of the radiation will be muchwider than any individual cell. Consequently, the beam of radiation neednot be separately targeted to a particular point on a cell or cellsurface to be effective, but can be directed to the general area of acell population without losing effectiveness. As a result, sensitivityto beam steering accuracy is reduced and throughput is dramaticallyincreased.

[0091] Fourth, the energy delivered by the pulse of radiation can belimited to at most 2, 1.5, 1, 0.7, 0.5, 0.3, 0.2, 0.1, 0.05, 0.02, 0.01or even 0.005 μJ/μm². This has the advantage of increasing the survivalrate while maintaining efficient loading rates. Moreover, unlikeprevious methods, the effective energy levels are low enough to allowthe use of common plastic specimen containers without damaging thecontainer.

[0092] The general method can also be modified to increase throughput.At the most basic level, the direction of the pulse of radiation can beadjusted to irradiate a second target cell in the population in a givenframe. Similarly, subsequent fields of view of the population of cellscan be processed as described above. This is especially useful when thepopulation of cells remains in a substantially stationary locationrelative to the lens. Alternatively, the cells can be moved relative tothe lens between applications of the method for further steps ofdetecting, locating and irradiating cells.

[0093] To maximize throughput of the cells, one or more of the steps ofthe method can be automated, as exemplified by the apparatus describedin detail above. For example, each of the steps can be controlled by amicroprocessor. Similarly, a static representation can be processed asan image or a data set stored in computer memory. By automating each ofthe steps, the optoinjection method can irradiate at least 5,000,10,000, 20,000, 50,000, 70,000, 100,000 or even 150,000 cells per minute

[0094] The following examples illustrate the use of the described methodand apparatus in different applications.

EXAMPLE 1 Autologous HSC Transplantation

[0095] A patient with a B cell-derived metastatic tumor in need of anautologous HSC transplant is identified by a physician. As a first stepin the treatment, the patient undergoes a standard HSC harvestprocedure, resulting in collection of approximately 1×10¹⁰ hematopoieticcells with an unknown number of contaminating tumor cells. The harvestedcells are enriched for HSC by a commercial immunoaffinity column (ISOLEX300, Nexell Therapeutics, Irvine, Calif.) that selects for cells bearingthe CD34 surface antigen, resulting in a population of approximately3×10⁸ hematopoietic cells, with an unknown number of tumor cells. Themixed population is thereafter contacted with anti-B cell antibodies(directed against CD20 and CD22) that are conjugated to phycoerythrin.The labeled antibodies specifically bind to the B cell-derived tumorcells.

[0096] The mixed cell population is then placed in a sterile specimencontainer on a substantially flat surface near confluence, atapproximately 500,000 cells per square centimeter. The specimen isplaced on the movable stage of the apparatus described above, and alldetectable tumor cells are identified by reference to phycoerythrin andtargeted with a lethal dose of energy from a treatment laser. The designof the apparatus allows the processing of a clinical-scale transplantspecimen in under 4 hours. The cells are recovered from the specimencontainer, washed, and then cryopreserved. Before the cells arereinfused, the patient is given high-dose chemotherapy to destroy thetumor cells in the patient's body. Following this treatment, theprocessed cells are thawed at 37° C. and are given to the patientintravenously. The patient subsequently recovers with no remission ofthe original cancer.

EXAMPLE 2 Allogeneic HSC Transplantation

[0097] In another embodiment, the significant risk and severity ofgraft-versus-host disease in the allogeneic HSC transplant setting canbe combated. A patient is selected for an allogeneic transplant once asuitable donor is found. Cells are harvested from the selected donor asdescribed in the above example. In this case, the cell mixture iscontacted with phycoerythrin-labeled anti-CD3 T-cell antibodies.Alternatively, specific allo-reactive T-cell subsets could be labeledusing an activated T-cell marker (e.g. CD69) in the presence ofallo-antigen. The cell population is processed by the apparatusdescribed herein, thereby precisely defining and controlling the numberof T-cells given to the patient. This type of control is advantageous,because administration of too many T-cells increases the risk ofgraft-versus-host disease, whereas too few T-cells increases the risk ofgraft failure and the risk of losing of the known beneficialgraft-versus-leukemia effect. The present invention and methods arecapable of precisely controlling the number of T-cells in an allogeneictransplant.

EXAMPLE 3 Tissue Engineering

[0098] In another application, the present apparatus is used to removecontaminating cells in inocula for tissue engineering applications. Cellcontamination problems exist in the establishment of primary cellcultures required for implementation of tissue engineering applications,as described by Langer and Vacanti, Tissue engineering: The challengesahead, Sci. Am. 280:86-89 (1999). In particular, chondrocyte therapiesfor cartilage defects are hampered by impurities in the cell populationsderived from cartilage biopsies. Accordingly, the present invention isused to specifically remove these types of cells from the inocula.

[0099] For example, a cartilage biopsy is taken from a patient in needof cartilage replacement. The specimen is then grown under conventionalconditions (Brittberg et al., Treatment of deep cartilage defects in theknee with autologous chondrocyte transplantation, N.E. J. Med.331:889-895 (1994)). The culture is then stained with a specific labelfor any contaminating cells, such as fast-growing fibroblasts. The cellmixture is then placed within the apparatus described and the labeled,contaminating cells are targeted by the treatment laser, therebyallowing the slower growing chondrocytes to fully develop in culture.

EXAMPLE 4 Stem Cell Therapy

[0100] Yet another embodiment involves the use of embryonic stem cellsto treat a wide variety of diseases. Since embryonic stem cells areundifferentiated, they can be used to generate many types of tissue thatwould find use in transplantation, such as cardiomyocytes and neurons.However, undifferentiated embryonic stem cells that are implanted canalso lead to a jumble of cell types which form a type of tumor known asa teratoma (Pedersen, R. A., Embryonic stem cells for medicine, Sci.Amer. 280:68-73 (1999)). Therefore, therapeutic use of tissues derivedfrom embryonic stem cells must include rigorous purification of cells toensure that only sufficiently differentiated cells are implanted. Theapparatus described herein is used to eliminate undifferentiated stemcells prior to implantation of embryonic stem cell-derived tissue in thepatient.

EXAMPLE 5 Generation of Human Tumor Cell Cultures

[0101] In another embodiment, a tumor biopsy is removed from a cancerpatient for the purpose of initiating a culture of human tumor cells.However, the in vitro establishment of primary human tumor cell culturesfrom manv tumor types is complicated by the presence of contaminatingprimary cell populations that have superior in vitro growthcharacteristics over tumor cells. For example, contaminating fibroblastsrepresent a major challenge in establishing many cancer cell cultures.The disclosed apparatus is used to particularly label and destroy thecontaminating cells, while leaving the biopsied tumor cells intact.Accordingly, the more aggressive primary cells will not overtake anddestroy the cancer cell line.

EXAMPLE 6 Generation of a Specific mRNA Expression Library

[0102] The specific expression pattern of genes within different cellpopulations is of great interest to many researchers, and many studieshave been performed to isolate and create libraries of expressed genesfor different cell types. For example, knowing which genes are expressedin tumor cells versus normal cells is of great potential value (Cossman,et al., Reed-Stemberg cell genome expression supports a B-cell lineage,Blood 94:411-416 (1999)). Due to the amplification methods used togenerate such libraries (e.g. PCR), even a small number of contaminatingcells will result in an inaccurate expression library (Cossman et al.,supra; Schutze and Lahr, Identification of expressed genes bylaser-mediated manipulation of single cells, Nature Biotechnol.16:737-742 (1998)). One approach to overcome this problem is the use oflaser capture microdissection (LCM), in which a single cell is used toprovide the starting genetic material for amplification (Schutze andLahr, supra). Unfortunately, gene expression in single cells is somewhatstochastic, and may be biased by the specific state of that individualcell at the time of analysis (Cossman et al., supra). Therefore,accurate purification of a significant cell number prior to extractionof mRNA would enable the generation of a highly accurate expressionlibrary, one that is representative of the cell population beingstudied, without biases due to single cell expression or expression bycontaminating cells. The methods and apparatus described in thisinvention can be used to purify cell populations so that nocontaminating cells are present during an RNA extraction procedure.

EXAMPLE 7 Transfection of a Specific Cell Population

[0103] Many research and clinical gene therapy applications are hamperedby the inability to transfect an adecuate number of a desired cell typewithout transfecting other cells that are present. The method of thepresent invention would allow selective targeting of cells to betransfected within a mixture of cells. By generating a photomechanicalshock wave at or near a cell membrane with a targeted energy source, atransient pore can be formed, through which genetic (or other) materialcan enter the cell. This method of gene transfer has been calledoptoporation (Palumbo et al. supra). The apparatus described above canachieve selective optoporation on only the cells of interest in a rapid,automated, targeted manner.

[0104] For example, white blood cells are plated in a specimen containerhaving a solution containing DNA to be transfected.Fluorescently-labeled antibodies having specificity for stem cells areadded into the medium and bind to the stem cells. The specimen containeris placed within the cell processing apparatus and a treatment laser istargeted to any cells that become fluorescent under the illuminationlaser light. The treatment laser facilitates transfection of DNAspecifically into the targeted cells.

EXAMPLE 8 Selection of Desirable Clones in a Biotechnology Application

[0105] In many biotechnology processes where cell lines are used togenerate a valuable product, it is desirable to derive clones that arevery efficient in producing the product. This selection of clones isoften carried out manually, by inspecting a large number of clones thathave been isolated in some manner. The present invention would allowrapid, automated inspection and selection of desirable clones forproduction of a particular product. For example, hybridoma cells thatare producing the greatest amounts of antibody can be identified by afluorescent label directed against the Fc region. Cells with no or dimfluorescent labeling are targeted by the treatment laser for killing,leaving behind the best producing clones for use in antibody production.

EXAMPLE 9 Automated Monitoring of Cellular Responses

[0106] Automated monitoring of cellular responses to specific stimuli isof great interest in high-throughput drug screening. Often, a cellpopulation in one well of a well-plate is exposed to a stimulus, and afluorescent signal is then captured over time from the cell populationas a whole. Using the methods and apparatus described herein, moredetailed monitoring could be done at the single cell level. For example,a cell population can be labeled to identify a characteristic of asubpopulation of cells that are of interest. This label is then excitedby the illumination laser to identify those cells. Thereafter, thetreatment laser is targeted at the individual cells identified by thefirst label, for the purpose of exciting a second label, therebyproviding information about each cell's response. Since the cells aresubstantially stationary on a surface, each cell could be evaluatedmultiple times, thereby providing temporal information about thekinetics of each cell's response. Also, through the use of the largearea scanning lens and galvanometer mirrors, a relatively large numberof wells could be quickly monitored over a short period of time.

[0107] As a specific example, consider the case of alloreactive T-cellsas presented in Example 2, above. In the presence of allo-antigen,activated donor T-cells could be identified by CD69. Instead of usingthe treatment laser to target and kill these cells, the treatment lasercould be used to examine the intracellular pH of every activated T-cellthrough the excitation and emitted fluorescence of carboxyfluoresceindiacetate. The targeted laser allows the examination of only cells thatare activated, whereas most screening methods evaluate the response ofan entire cell population. If a series of such wells are being monitoredin parallel, various agents could be added to individual wells, and thespecific activated T-cell response to each agent could be monitored overtime. Such an apparatus would provide a high-throughput screening methodfor agents that ameliorate the alloreactive T-cell response ingraft-versus-host disease. Based on this example, one skilled in the artcould imagine many other examples in which a cellular response to astimulus is monitored on an individual cell basis, focusing only oncells of interest identified by the first label.

EXAMPLE 10 Photobleaching Studies

[0108] Photobleaching, and/or photobleach recovery, of a specific areaof a fluorescently-stained biological sample is a common method that isused to assess various biological processes. For example, a cellsuspension is labeled with rhodamine 123, which fluorescently stainsmitochondria within the cells. Using the instant illumination laser, themitochondria within one or more cells are visualized due to rhodamine123 fluorescence. The treatment laser is then used to deliver a focusedbeam of light that results in photobleaching of the rhodamine 123 in asmall area within one or more cells. The photobleached area(s) thenappear dark immediately thereafter, whereas adjacent areas areunaffected. A series of images are then taken using the illuminationlaser, providing a time-lapse series of images that document themigration of unbleached mitochondria into the area that wasphotobleached with the treatment laser. This approach can be used toassess the motion, turnover, or replenishment of many biologicalstructures within cells.

[0109] Thus, in cultured rat neurites, the photobleach recovery ofmitochondria is a measure of the size of the mobile pool of mitochondriawithin each cell (Chute, et al.,Analysis of the steady-state dynamicsorganelle motion in cultured neurites, Clin. Exp. Pharmco. Physiol.22:360 (1995)). The rate of photobleach recovery in these cells isdependent on intracellular calcium and magnesium concentrations, energystatus, and microtubule integrity. Neurotoxic substances, such as taxolor vinblastine, will affect the rate of photobleach recovery. Therefore,an assay for neurotoxic substances could be based on the measurement ofphotobleach recovery of mitochondria within a statistically significantnumber of neurites that had been exposed to various agents in the wellsof a multi-well plate. In such an application, the apparatus describedherein and used as described above, would provide a rapid automatedmethod to assess neurotoxicity of many substances on a large number ofcells. Based on this example, one skilled in the art could imagine manyother examples in which photobleaching is induced and photobleachrecovery is monitored in order to obtain useful information from abiological specimen.

EXAMPLE 11 Uncaging Studies

[0110] Use of caged compounds to study rapid biological processesinvolves the binding (i.e. caging) of a biologically relevant substancein an inactive state, allowing the caged substance to diffuse into thebiological specimen (a relatively slow process), and then using a laserto induce a photolysis reaction (a relatively fast process) whichliberates (i.e. uncages) the substance in situ over microsecond timescales. The biological specimen is then observed in short time-lapsemicroscopy in order to determine the effect of the uncaged substance onsome biological process. Cages for many important substances have beendescribed, including Dioxygen, cyclic ADP ribose (cADPR), nicotinic acidadenine dinucleotide phosphate (NAADP), nitric oxide (NO), calcium,L-aspartate, and adenosine triphosphate (ATP). Chemotaxis is one exampleof a physiological characteristic that can be studied by uncagingcompounds.

[0111] Uncaging studies involve the irradiation of a portion of abiological specimen with laser light followed by examination of thespecimen with time-lapse microscopy. The apparatus of the currentinvention has clear utility in such studies. As a specific example,consider the study of E. coli chemotaxis towards L-aspartate (Jasuja etal., Chemotactic responses of Escherichia coli to small jumps ofphotoreleased L-aspartate, Biophys. J. 76:1706 (1999)). Thebeta-2,6-dinitrobenzyl ester of L-aspartic acid and the1-(2-nitrophenyl)ethyl ether of 8-hydroxylpyrene-1,3,6-tris-sulfonicacid are added to the wells of a well plate containing E. coli. Uponirradiation with the treatment laser, a localized uncaging ofL-aspartate and the fluorophore 8-hydroxylpyrene-1,3,6-tris-sulfonicacid (pyranine) is induced. The L-aspartate acts as a chemoattractantfor E. coli., and in subsequent fluorescent images (using theillumination laser) the pyranine fluorophore acts as an indicator of thedegree of uncaging that has occurred in the local area of irradiation.Time-lapse images of the E. coli. in the vicinity illuminated by visiblewavelength light, such as from the back-light, of the uncaging event areused to measure the chemotactic response of the microorganisms to thelocally uncaged L-aspartate. Due to the nature of the present invention,a large number of wells, each with a potential anti-microbial agentadded, are screened in rapid order to determine the chemotactic responseof microorganisms. Based on this example, one skilled in the art couldimagine many other examples in which uncaging is induced by thetreatment laser, followed by time-lapse microscopy in order to obtainuseful information on a large number of samples in an automated fashion.

EXAMPLE 12 Optoinjection of NIH-3T3 Cells with 70 kD Dextran

[0112] This example illustrates an optoinjection method for transientlypermeabilizing a target cell. NIH-3T3 cells were grown in a 96-wellplate. The growth medium was removed and replaced with PBS containing 1%BSA and 0.1 mM Texas-Red-Dextran (70 kDa) (Molecular Probes, Eugene,Oreg.). Upon illumination of the cells under broad-spectrum light, astatic image (FIG. 8A) was obtained to determine which cells to target.

[0113] A 30 micron energy beam having a wavelength of 523 nm wasdirected sequentially to the target cells through a flat-field lenshaving a magnification of 2.5×, a numerical aperture (N.A.) of 0.25, anda working distance of greater than 10 mm. Over 500 cells were targetedper second.

[0114] After irradiating the target cells, the wells were washed, andSytox Blue (10 mM, Molecular Probes) was added to stain non-viablecells. As shown in FIG. 8B, about 70% of the cells showed loading of theTexas-Red-Dextran as an exogenous molecule. Moreover, only one cell wasnon-viable (FIG. 8C), equivalent to about a 95% survival rate.

EXAMPLE 13 Optoinjection of SU-DHL-4 Cells with Sytox Green

[0115] Using the same hardware apparatus as in Example 12, SU-DHL-4cells were placed in 96-well plates in PBS with 1% HSA. Themembrane-impermeable dye Sytox Green (Molecular Probes) was added at0.05 mM, the cells were allowed to settle, and then were imaged andtargeted with a 30 micron laser beam. Different energy levels rangingfrom 2 to 15 μJ per pulse of the laser were used in each of five wells,with each target cell receiving one pulse. As show in FIG. 9, theefficiency of optoinjection was energy dose-dependent, ranging from 58%at 4 μJ/cell (0.0057 μJ/μm²) to 92% at 15 μJ/cell (0.021 μJ/μm²). In allcases, cell viability was greater than 95%.

EXAMPLE 14 Optoinjection of 293T Cells with pEGFP-N1 Plasmid

[0116] In this experiment, the exogenous molecule was a DNA plasmid of4.3 kb encoding the fluorescent EGFP protein (pEGFP-N1). The samehardware apparatus was used as in Example 12. The cells were in a mediumof PBS and 1% HSA, and then 0.1 microgram of plasmid was added to eachwell. The cells were imaged, located and targeted with the laser beamsuch that each cell received 1 to 8 pulses of 15 μJ each (0.021 μJ/μm²).The cells were washed, placed in growth medium, and then cultured for 48to 96 hours. After culturing, cells were evaluated for the expression ofthe fluorescent EGFP protein. As shown in FIG. 10, a number of cellsdisplayed the fluorescent phenotype in the treated wells (FIG. 10A),whereas no fluorescence was observed in the control well (FIG. 10B),which were treated identically with the exception of delivering thelaser pulses.

[0117] Although aspects of the present invention have been described byparticular embodiments exemplified herein, the present invention is notso limited. The present invention is only limited by the claims appendedbelow.

We claim:
 1. A method for transiently permeabilizing a target cell,comprising the steps of (a) illuminating a population of substantiallystationary cells contained in a frame; (b) obtaining a staticrepresentation of at least one property of light directed simultaneouslyfrom the frame; (c) locating a target cell in the population of cells,wherein the target cell is located with reference to the staticrepresentation; and (d) irradiating the target cell with a pulse ofradiation; whereby the target cell is transiently permeabilized.
 2. Amethod for transiently permeabilizing a target cell, comprising thesteps of (a) illuminating a population of cells contained in a frame,wherein the cells are illuminated through a lens having a numericalaperture of at most 0.5; (b) detecting at least one property of lightdirected from the frame and through the lens; (c) locating a target cellin the population of cells, wherein the target cell is located withreference to the detected property of light; and (d) irradiating thetarget cell with a pulse of radiation; whereby the target cell istransiently permeabilized.
 3. A method for transiently permeabilizing atarget cell, comprising the steps of (a) illuminating a population ofcells contained in a frame; (b) detecting at least one property of lightdirected from the frame; (c) locating a target cell in the population ofcells, wherein the target cell is located with reference to the detectedproperty of light; and (d) irradiating the target cell with a pulse ofradiation, wherein the pulse of radiation has a diameter of at least 5microns at the point of contact with the target cell; whereby the targetcell is transiently permeabilized.
 4. A method for transientlypermeabilizing a target cell, comprising the steps of (a) illuminating apopulation of cells contained in a frame; (b) detecting at least oneproperty of light directed from the frame; (c) locating a target cell inthe population of cells, wherein the target cell is located withreference to the detected property of light; and (d) irradiating thetarget cell with a pulse of radiation, wherein the pulse of radiationdelivers at most 1 μJ/μm²; whereby the target cell is transientlypermeabilized.
 5. The method of claim 2, 3 or 4, wherein the populationof cells is substantially stationary.
 6. The method of claim 2, 3 or 4,wherein the property of light detected in step (b) is obtained as astatic representation of light transmitted simultaneously from theframe, whereby the target cell located in step (c) is located withreference to the static representation.
 7. The method of claim 2, 3 or4, wherein step (b) further comprises obtaining a static representationof the at least one property of light transmitted simultaneously fromthe frame, whereby step (c) further comprises locating the target cellwith reference to the static representation.
 8. The method of claim 1,2, 3 or 4, wherein at least one property of light is fluorescence andthe target cell is located with reference to the fluorescence.
 9. Themethod of claim 1, 3 or 4, wherein the population of cells isilluminated through a lens having numerical aperture of at most 0.5 andthe target cell is located with reference to a property of lightdirected from the frame and through the lens.
 10. The method of claim 2,wherein the lens has a numerical aperture of at most 0.4.
 11. The methodof claim 2, wherein the lens as a numerical aperture of at most 0.3. 12.The method of claim 2, wherein the lens has flat field correction. 13.The method of claim 2, wherein the lens has a working distance of atleast 5 mm.
 14. The method of claim 2, wherein the lens has a workingdistance of at least 10 mm.
 15. The method of claim 1, 2 or 4, whereinthe pulse of radiation has a diameter of at least 5 microns at the pointof contact with the target cell.
 16. The method of claim 3, wherein thepulse of radiation has a diameter of at least 7 microns at the point ofcontact with the target cell.
 17. The method of claim 3, wherein thepulse of radiation has a diameter of at least 10 microns at the point ofcontact with the target cell.
 18. The method of claim 3, wherein thepulse of radiation has a diameter of at least 20 microns at the point ofcontact with the target cell.
 19. The method of claim 1, 2 or 3, whereinthe pulse of radiation delivers at most 1 μJ/μm².
 20. The method ofclaim 4, wherein the pulse of radiation delivers at most 0.1 μJ/μm². 21.The method of claim 4, wherein the pulse of radiation delivers at most0.01 μJ/μm².
 22. The method of claim 1, further comprising the step of(e) adjusting the direction of the pulse of radiation to irradiate asecond target cell in the population, whereby the second target cell istransiently permeabilized.
 23. The method of claim 1, wherein the framehas an area of at least 50 mm².
 24. The method of claim 1, wherein theframe has an area of at least 85 mm².
 25. The method of claim 1, whereinthe frame has an area of at least 115 mm².
 26. The method of claim 1,wherein the population of cells is illuminated with a laser.
 27. Themethod of claim 1, wherein the population of cells is illuminated with alamp.
 28. The method of claim 1, wherein the population of cells isilluminated with light selected from the group consisting of visible,ultraviolet, and infrared wavelengths.
 29. The method of claim 1,wherein the property of light is selected from the group consisting ofvisible, ultraviolet, or infrared wavelengths.
 30. The method of claim1, wherein the property of light is transmittance and the target cell islocated with reference to the transmittance.
 31. The method of claim 1,wherein the property of light is polarization and the target cell islocated with reference to the polarization.
 32. The method of claim 1,wherein the property of light is reflectance and the target cell islocated with reference to the reflectance.
 33. The method of claim 1,wherein the property of light is phase contrast illumination and thetarget cell is located with reference to the phase contrastillumination.
 34. The method of claim 1, wherein the property of lightis intensity and the target cell is located with reference to theintensity.
 35. The method of claim 1, wherein greater than 50% of theirradiated target cells are viable after the method is performed. 36.The method of claim 1, wherein greater than 80% of the irradiated targetcells are viable after the method is performed.
 37. The method of claim1, wherein greater than 90% of the irradiated target cells are viableafter the method is performed.
 38. The method of claim 1, wherein thetarget cell is a procaryotic cell.
 39. The method of claim 1, whereinthe target cell is a eucaryotic cell.
 40. The method of claim 1, whereinthe target cell is selected from the group consisting of an animal cell,plant cell, yeast cell, human cell and non-human primate cell.
 41. Themethod of claim 1, wherein the population of cells contains cellsassociated with an exogenous label.
 42. The method of claim 41, whereinthe label is a fluorophore.
 43. The method of claim 1, wherein thetarget cell is associated with an exogenous label.
 44. The method ofclaim 1, wherein the target cell is in the presence of an exogenousmolecule, whereby the exogenous molecule enters the transientlypermeabilized cell.
 45. The method of claim 44, wherein the exogenousmolecule is selected from the group consisting of a nucleic acid,polypeptide, carbohydrate, lipid, and small molecule.
 46. The method ofclaim 45, wherein the small molecule is a dye capable of absorbingvisible, ultraviolet or infrared light.
 47. The method of claim 44,wherein the molecular weight of the exogenous molecule is greater than0.1 kiloDalton.
 48. The method of claim 44, wherein the molecular weightof the exogenous molecule is greater than 0.3 kiloDalton.
 49. The methodof claim 44, wherein the molecular weight of the exogenous molecule isgreater than 1 kiloDalton.
 50. The method of claim 44, wherein themolecular weight of the exogenous molecule is greater than 3kiloDaltons.
 51. The method of claim 44, wherein the molecular weight ofthe exogenous molecule is greater than 10 kiloDaltons.
 52. The method ofclaim 44, wherein the molecular weight of the exogenous molecule isgreater than 30 kiloDaltons.
 53. The method of claim 44, wherein themolecular weight of the exogenous molecule is greater than 70kiloDaltons.
 54. The method of claim 1, wherein step (b) furthercomprises obtaining a second static representation of at least oneproperty of light directed simultaneously from the frame.
 55. The methodof claim 54, wherein the target cell is located with reference to saidfirst and second static representation.
 56. The method of claim 1,wherein steps (c) and (d) are repeated so that more than one target cellis located and irradiated.
 57. The method of claim 1, further comprisingthe steps of (e) illuminating a population of cells contained in asecond frame (f) obtaining a static representation of at least oneproperty of light directed from the second frame and through the lens,and repeating steps (c) through (d).
 58. The method of claim 57, whereinthe population of cells remains in a substantially stationary locationrelative to the lens.
 59. The method of claim 57, wherein at least10,000 cells are irradiated per minute.
 60. The method of claim 57,wherein at least 20,000 cells are irradiated per minute.
 61. The methodof claim 57, wherein at least 50,000 cells are irradiated per minute.62. The method of claim 57, wherein at least 100,000 cells areirradiated per minute.
 63. The method of claim 57, further comprisingthe step of (g) moving the population of cells relative to the lens andrepeating steps (a) through (f).
 64. The method of claim 57, whereinsteps (a) through (f) are automated.
 65. The method of claim 1, furthercomprising the steps of (e) moving the population of cells relative tothe lens and repeating steps (a) through (d).
 66. The method of claim 1,wherein steps (a) through (d) are automated.
 67. The method of claim 1,wherein the static representation comprises an image.
 68. The method ofclaim 1, wherein the static representation comprises a set of datastored in computer memory.
 69. The method of claim 1, further comprisinga camera having a magnification between 2× and 40×.
 70. The method ofclaim 1, further comprising a camera having a magnification between 2.5×and 25×.